PROTEIN INTERACTION ANALYSIS CROSSLINKING AND LABEL TRANSFER crosslinking protein interaction analysis Under physiological condi�on, most protein-protein interac�ons are transient, and happen in a very short dura�on, increasing the difficul�es to study them. Crosslinking reagents, or crosslinkers, provide the analy�cal solu�on to capture protein-protein complexes by covalently binding them together as they interact and freezing even transient, weak interac�ons for consequent isola�on and characteriza�on. O N
O
NaO3S
O
O
O
O
O
Reac�ve group (variable)
Spacer
N
SO3Na
O
Reac�ve group (variable)
Select cross-linker
Reac�on
Protein A
Interprotein cross-link Protein X Intraprotein cross-link
Protein B Protein Y Targeted Interac�ons
Diges�on
SDS gel
Large-scale Interac�ons
Interac�ons evidence
Diges�on Linear pep�des
Cross-linked pep�des
Intensity
LC-ESI-MS
LC-ESI-MS
m/z
Intensity
Database search
m/z m/z
Database search
m/z
m/z
label transfer protein interaction analysis The label transfer approach incorporates crosslinking methodology to study protein-protein interac�ons by labeling proteins that interact with a protein of interest. This method can be used to discover novel interac�ons, confirm puta�ve interac�ons suggested by other techniques, and inves�gate protein complexes. In addi�on, the label transfer method can detect weak or transient protein interac�ons that o�en fail to detect by co-immunoprecipita�on, or pull-down method. L PR
L
O
PR
O NHS-SO3-
S S (CH2)n
Interac�on of labeled bait protein with prey protein in vitro
O S
N
H
Protein 1
S
L
Protein 1
Protein 2
Reac�ng decoy proteins to label transfer reagent (LTR)
(CH2)n
NH
S S (CH2)n
NH2
Protein 1
Ac�vate photoreac�ve groups on the crosslinker
O
UV L PR
O S S (CH2)n
NH
Protein 2
Protein 1 Protein 2
PR
PR
b3
HS
y2
L b2
Cut the crosslinker spacer to release a�ached bait proteins for label transfer
y3
MALDI/ESI-CID/HCD/ETD
Protein 2
m/z
m/z
Mass spectrometry analysis
characteristics Protein Crosslinking
1 2
Label Transfer
Ability to detect weak or transient protein interac�ons In vivo cross-linking: interac�ons between proteins can be captured in their natural environment. However, this method cannot strictly control the reac�on condi�ons. If mul�ple proteins have func�onal groups that specifically react with the cross-linking agent, the cross-linking agent will react with the mul�ple proteins.
3
In vitro cross-linking: strictly control more reac�on condi�ons for specific cross-linking reac�on. But lack of physiological condi�ons.
4
Mul�ple protein cross-linking methods, including chemical cross-linking, enzyma�c cross-linking, and photoprotein cross-linking
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1
Ability to detect weak or transient protein interac�ons
2
Can be used to purify and detect prey proteins
3
Most LTRs can directly label specific protein domains, but they are generally not suitable for mapping protein-protein interac�on networks because homing molecules a�ached to cross-linking agents usually only direct cross-linking to the binding domain.